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Jun Kinase Delays Caspase-9 Activation by Interaction with the Apoptosome*

  • Author Footnotes
    1 Predoctoral fellow of the American Heart Association, Florida Affiliate.
    Thanh H. Tran
    Footnotes
    1 Predoctoral fellow of the American Heart Association, Florida Affiliate.
    Affiliations
    Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136
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  • Author Footnotes
    2 Present address: Gottsegen Hungarian Institute of Cardiology, 29 Haller Str., H-1096 Budapest, Hungary. Postdoctoral fellow of the American Heart Association, Puerto Rico Affiliate, and recipient of a research award from the George Soros Foundation.
    Peter Andreka
    Footnotes
    2 Present address: Gottsegen Hungarian Institute of Cardiology, 29 Haller Str., H-1096 Budapest, Hungary. Postdoctoral fellow of the American Heart Association, Puerto Rico Affiliate, and recipient of a research award from the George Soros Foundation.
    Affiliations
    Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136
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  • Author Footnotes
    3 Supported by a National Institutes of Health training grant postdoctoral fellowship.
    Claudia O. Rodrigues
    Footnotes
    3 Supported by a National Institutes of Health training grant postdoctoral fellowship.
    Affiliations
    Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136
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  • Keith A. Webster
    Affiliations
    Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136
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  • Nanette H. Bishopric
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular and Cellular Pharmacology (R-189), P. O. Box 016189, Miami, FL 33101. Tel.: 305-243-6775; Fax: 305-243-6082
    Affiliations
    Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136

    Department of Pediatrics, University of Miami Miller School of Medicine, Miami, Florida 33136

    Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida 33136
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  • Author Footnotes
    * This work was supported in part by NHLBI National Institutes of Health Grants R-01-HL71094 (to N. H. B.) and R-01-HL44578 (to K. A. W.) the Fondation Leducq (to N. H. B.) and the Florida Heart Research Institute (to N. H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
    1 Predoctoral fellow of the American Heart Association, Florida Affiliate.
    2 Present address: Gottsegen Hungarian Institute of Cardiology, 29 Haller Str., H-1096 Budapest, Hungary. Postdoctoral fellow of the American Heart Association, Puerto Rico Affiliate, and recipient of a research award from the George Soros Foundation.
    3 Supported by a National Institutes of Health training grant postdoctoral fellowship.
Open AccessPublished:May 04, 2007DOI:https://doi.org/10.1074/jbc.M702210200
      Activation of c-Jun N-terminal kinase 1/2 (JNK) can delay oxidant-induced cell death, but the mechanism is unknown. We found that oxidant stress of cardiac myocytes activated both JNK and mitochondria-dependent apoptosis and that expression of JNK inhibitory mutants accelerated multiple steps in this pathway, including the cleavage and activation of caspases-3 and -9 and DNA internucleosomal cleavage, without affecting the rate of cytochrome c release; JNK inhibition also increased caspase-3 and -9 cleavage in a cell-free system. On activation by GSNO or H2O2, JNK formed a stable association with oligomeric Apaf-1 in a ∼1.4–2.0 mDa pre-apoptosome complex. Formation of this complex could be triggered by addition of cytochrome c and ATP to the cell-free cytosol. JNK inhibition abrogated JNK-Apaf-1 association and accelerated the association of procaspase-9 and Apaf-1 in both intact cells and cell-free extracts. We conclude that oxidant-activated JNK associates with Apaf-1 and cytochrome c in a catalytically inactive complex. We propose that this interaction delays formation of the active apoptosome, promoting cell survival during short bursts of oxidative stress.
      Both permanent and transient interruptions of blood flow to the heart cause oxidative stress (
      • Duranteau J.
      • Chandel N.S.
      • Kulisz A.
      • Shao Z.
      • Schumacker P.T.
      ,
      • Vanden Hoek T.L.
      • Becker L.B.
      • Shao Z.
      • Li C.
      • Schumacker P.T.
      ,
      • Kevin L.G.
      • Camara A.K.
      • Riess M.L.
      • Novalija E.
      • Stowe D.F.
      ,
      • Ueda R.
      • Konno N.
      • Nakatani M.
      • Katagiri T.
      ). During hypoxia, and during restoration of oxygen, the production of reactive oxygen species from both intra- and extracellular sources increases significantly, including varying proportions of superoxide, hydrogen peroxide, hydroxyl radical, and nitric oxide (
      • Borutaite V.
      • Brown G.C.
      ,
      • Han H.
      • Long H.
      • Wang H.
      • Wang J.
      • Zhang Y.
      • Wang Z.
      ). Oxidant stress in turn causes myocyte damage and loss, either by direct oxidation of cellular components, or by activation of one or more pathways of programmed cell death (
      • Bishopric N.H.
      • Andreka P.
      • Slepak T.
      • Webster K.A.
      ,
      • Czerski L.
      • Nunez G.
      ). The intrinsic, or mitochondrial, pathway has been implicated in cardiac myocyte death induced by glucose or serum deprivation (
      • Bialik S.
      • Cryns V.L.
      • Drincic A.
      • Miyata S.
      • Wollowick A.L.
      • Srinivasan A.
      • Kitsis R.N.
      ), overexpression of Gαq (
      • Adams J.W.
      • Pagel A.L.
      • Means C.
      • Oxsenberg D.
      • Armstrong R.C.
      • Brown J.H.
      ) and oxidant stress (
      • Aoki H.
      • Kang P.M.
      • Hampe J.
      • Yoshimura K.
      • Noma T.
      • Matzuzaki M.
      • Izumo S.
      ). This pathway is initiated by release of cytochrome c through the outer mitochondrial membrane into the cytosol that catalyzes formation of the apoptosome, a multiprotein complex required for activation of procaspase-9 and effector caspase-dependent cell death (
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Li P.
      • Nijhawan D.
      • Budihardjo I.
      • Srinivasula S.M.
      • Ahmad M.
      • Alnemri E.S.
      • Wang X.
      ,
      • Acehan D.
      • Jiang X.
      • Morgan D.G.
      • Heuser J.E.
      • Wang X.
      • Akey C.W.
      ). However, the conditions governing formation and composition of the apoptosome in long-lived, essentially non-proliferative cells such as cardiac myocytes remain undefined (
      • Czerski L.
      • Nunez G.
      ).
      The c-Jun N-terminal kinases (JNKs 1, 2, and 3)
      The abbreviations used are: JNK, c-Jun N-terminal kinase; XIAP, X-linked inhibitor of apoptosis; TNF, tumor necrosis factor; GSNO, S-nitrosoglutathione; GFP, green fluorescent protein; ERK, extracellular-signal regulated kinase; JIA, inactive JNK; Ad-dnJNK, adenovirus expressing dominant negative JNK; HEK, human embryonic kidney.
      5The abbreviations used are: JNK, c-Jun N-terminal kinase; XIAP, X-linked inhibitor of apoptosis; TNF, tumor necrosis factor; GSNO, S-nitrosoglutathione; GFP, green fluorescent protein; ERK, extracellular-signal regulated kinase; JIA, inactive JNK; Ad-dnJNK, adenovirus expressing dominant negative JNK; HEK, human embryonic kidney.
      are serine/threonine kinases at the terminus of one of the three-component modules of the mitogen-activated protein (MAP) kinase family (,
      • Chang L.
      • Karin M.
      ) JNK isoforms play major roles in development, cell proliferation, cell differentiation, and apoptosis (
      • Kuan C.Y.
      • Yang D.D.
      • Samanta Roy D.R.
      • Davis R.J.
      • Rakic P.
      • Flavell R.A.
      ,
      • Sabapathy K.
      • Hu Y.
      • Kallunki T.
      • Schreiber M.
      • David J.P.
      • Jochum W.
      • Wagner E.F.
      • Karin M.
      ,
      • Dong C.
      • Yang D.D.
      • Tournier C.
      • Whitmarsh A.J.
      • Xu J.
      • Davis R.J.
      • Flavell R.A.
      ,
      • David J.P.
      • Sabapathy K.
      • Hoffmann O.
      • Idarraga M.H.
      • Wagner E.F.
      ,
      • Leppa S.
      • Bohmann D.
      ). JNK-1 and JNK-2 are abundant in myocardium and appear to be functionally redundant; JNK-3 is most abundant in brain, where it and JNK-1 play region-specific positive and negative roles in neuronal apoptosis (
      • Kuan C.Y.
      • Yang D.D.
      • Samanta Roy D.R.
      • Davis R.J.
      • Rakic P.
      • Flavell R.A.
      ,
      • Brecht S.
      • Kirchhof R.
      • Chromik A.
      • Willesen M.
      • Nicolaus T.
      • Raivich G.
      • Wessig J.
      • Waetzig V.
      • Goetz M.
      • Claussen M.
      • Pearse D.
      • Kuan C.Y.
      • Vaudano E.
      • Behrens A.
      • Wagner E.
      • Flavell R.A.
      • Davis R.J.
      • Herdegen T.
      ). Studies in mouse embryo fibroblasts suggest that JNK is required for the release of cytochrome c in response to UV irradiation (
      • Tournier C.
      • Hess P.
      • Yang D.D.
      • Xu J.
      • Turner T.K.
      • Nimnual A.
      • Bar-Sagi D.
      • Jones S.N.
      • Flavell R.A.
      • Davis R.J.
      ), but dispensable for TNF-α-induced apoptosis (
      • Lamb J.A.
      • Ventura J.J.
      • Hess P.
      • Flavell R.A.
      • Davis R.J.
      ). On the other hand, disruption of MKK4 and MKK7, which prevents JNK activation, had no impact on mitochondria-mediated apoptosis in embryonic stem cells (
      • Nishitai G.
      • Shimizu N.
      • Negishi T.
      • Kishimoto H.
      • Nakagawa K.
      • Kitagawa D.
      • Watanabe T.
      • Momose H.
      • Ohata S.
      • Tanemura S.
      • Asaka S.
      • Kubota J.
      • Saito R.
      • Yoshida H.
      • Mak T.W.
      • Wada T.
      • Penninger J.M.
      • Azuma N.
      • Nishina H.
      • Katada T.
      ).
      Oxidant stress strongly activates JNK1/2 in cardiac myocytes (
      • Laderoute K.R.
      • Webster K.A.
      ,
      • Clerk A.
      • Michael A.
      • Sugden P.H.
      ); however, the significance of this activation, whether in promoting or neutralizing reactive oxygen species-mediated apoptosis, remains unresolved, and the molecular targets of JNK in this context are unknown. Studies of JNK function in cardiac myocytes (
      • Minamino T.
      • Yujiri T.
      • Papst P.J.
      • Chan E.D.
      • Johnson G.L.
      • Terada N.
      ,
      • Andreka P.
      • Dougherty C.
      • Slepak T.I.
      • Webster K.A.
      • Bishopric N.H.
      ,
      • Hreniuk D.
      • Garay M.
      • Gaarde W.
      • Monia B.P.
      • McKay R.A.
      • Cioffi C.L.
      ,
      • Dougherty C.J.
      • Kubasiak L.A.
      • Prentice H.
      • Andreka P.
      • Bishopric N.H.
      • Webster K.A.
      ,
      • Ferrandi C.
      • Ballerio R.
      • Gaillard P.
      • Giachetti C.
      • Carboni S.
      • Vitte P.A.
      • Gotteland J.P.
      • Cirillo R.
      ,
      • Remondino A.
      • Kwon S.H.
      • Communal C.
      • Pimentel D.R.
      • Sawyer D.B.
      • Singh K.
      • Colucci W.S.
      ,
      • Engelbrecht A.M.
      • Niesler C.
      • Page C.
      • Lochner A.
      ) and in genetically manipulated mice have also produced conflicting results. Mice deficient in MEKK1, a JNK kinase kinase, displayed enhanced pressure overload-induced apoptosis, suggesting a protective role for JNK (
      • Sadoshima J.
      • Montagne O.
      • Wang Q.
      • Yang G.
      • Warden J.
      • Liu J.
      • Takagi G.
      • Karoor V.
      • Hong C.
      • Johnson G.L.
      • Vatner D.E.
      • Vatner S.F.
      ). In independent studies, sustained myocardial JNK activation via activated MKK7 caused cardiomyopathy but no increase in apoptosis (
      • Petrich B.G.
      • Eloff B.C.
      • Lerner D.L.
      • Kovacs A.
      • Saffitz J.E.
      • Rosenbaum D.S.
      • Wang Y.
      ,
      • Kaiser R.A.
      • Liang Q.
      • Bueno O.F.
      • Huang Y.
      • Lackey T.
      • Klevitsky R.
      • Hewett T.E.
      • Molkentin J.D.
      ). In the latter study, low-level JNK activation conferred protection from ischemia-reperfusion injury, but so did deletion of either JNK1 or JNK2, or expression of dominant negative JNK mutants (
      • Kaiser R.A.
      • Liang Q.
      • Bueno O.F.
      • Huang Y.
      • Lackey T.
      • Klevitsky R.
      • Hewett T.E.
      • Molkentin J.D.
      ). JNK actions thus appear to be highly context-dependent, and further clarification requires a better understanding of JNK substrates and partners in the apoptotic signaling cascade.
      We have previously shown that inhibition of JNK accelerates oxidant-mediated apoptosis in neonatal rat cardiac myocytes (
      • Andreka P.
      • Dougherty C.
      • Slepak T.I.
      • Webster K.A.
      • Bishopric N.H.
      ,
      • Dougherty C.J.
      • Kubasiak L.A.
      • Prentice H.
      • Andreka P.
      • Bishopric N.H.
      • Webster K.A.
      ). In the present study, we examined the effects and molecular targets of JNK in cardiac myocytes exposed to two distinct physiologic sources of oxidative stress, the nitric oxide donor S-nitrosoglutathione (GSNO) and hydrogen peroxide (H2O2). JNK inhibition in the presence of either stressor had no effect on cytochrome c release, but accelerated post-mitochondrial apoptotic events, including caspase-9 association with Apaf-1 and activation, both in intact myocytes and in a cell-free system. Oxidant-mediated JNK activation stimulated the formation of a JNK-Apaf-1 complex that excluded active caspase-9. This finding suggests a broad adaptive role for JNK activation in oxidant-rich environments such as the myocardium.

      EXPERIMENTAL PROCEDURES

      Antibodies—Polyclonal antibodies against caspase-9 and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against Apaf-1 were obtained from eBioscience (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, CA). The latter was also the source for antibodies against caspase-9, JNK (FL), JNK (F3), HA (F-7), normal rabbit and mouse IgGs.
      Primary Culture of Rat Neonatal Cardiomyocytes—All experiments were conducted under University of Miami Animal Care and Use Committee-approved protocols. Primary cultures of rat neonatal cardiomyocytes were generated as previously described (
      • Bishopric N.H.
      • Kedes L.
      ,
      • Ing D.J.
      • Zang J.
      • Dzau V.J.
      • Webster K.A.
      • Bishopric N.H.
      ).
      Recombinant Adenovirus Generation and Infection—Construction of the dominant negative JNK1 adenovirus has been previously described (
      • Dougherty C.J.
      • Kubasiak L.A.
      • Prentice H.
      • Andreka P.
      • Bishopric N.H.
      • Webster K.A.
      ). Adenoviruses expressing inactive JNK (JIA) and dominant negative casapase-9 were generated as described by He et al. (
      • He T.C.
      • Zhou S.
      • da Costa L.T.
      • Yu J.
      • Kinzler K.W.
      • Vogelstein B.
      ). The plasmid encoding a dominant negative mutant of rat caspase-9 (C327A) was the gift of Dr. James M. Angelastro (
      • Angelastro J.M.
      • Moon N.Y.
      • Liu D.X.
      • Yang A.S.
      • Greene L.A.
      • Franke T.F.
      ). The caspase-9 mutant gene was excised with KpnI and XbaI and inserted into the kanamycin-resistant pAdTrack-CMV shuttle vector (Microbix Biosystems Inc., Ontario, Canada). The plasmid encoding inactive JNK protein, a fusion between JNKK2 and JNK1 (APY) was the kind gift of Dr. Anning Lin (
      • Zheng C.
      • Xiang J.
      • Hunter T.
      • Lin A.
      ). All viral vectors were tagged with green fluorescent protein (GFP). Infection with adenoviral vectors was performed overnight at 5 multiplicity of infection and cells were incubated for a further 48 h. Infection efficiency and recombinant protein expression were monitored by GFP expression.
      Cell Fractionation and in Vitro Caspase Cleavage Analysis—Cell fractionation analysis was performed as described, with few modifications (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ). After treatment, cells were washed with ice-cold phosphate-buffered saline and harvested in cell fractionation buffer (CFB, pH 7.2): 20 mm HEPES-KOH, pH 7.5, 10 mm KCl, 1 mm EDTA pH 8.0, 1 mm EGTA, pH 8.0, 1.5 mm MgCl2, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride and proteinase inhibitors. Lysates were incubated on ice for 30 min, homogenized using 25 strokes of a pellet pestle, and centrifuged at 14,000 × g for 15 min at 4 °C. The supernatants (cytosolic fraction) were reserved, and the pellets were resuspended in 1× SDS sample buffer and boiled for 5 min at 100 °C to yield the mitochondrial fraction. Protein was quantitated using the BCA method (Pierce). For caspase cleavage analysis, cytosolic lysates (100 μg) in a final volume of 40 μl per reaction were incubated with or without cytochrome c (10 μm) and ATP (2 mm) at 37 °C for 3 h. Reactions were stopped by adding 4× SDS sample buffer and boiling for 5 min. Proteins were separated by 15% SDS-PAGE for immunoblot analysis.
      Immunoprecipitation—Proteins (100–200 μg) were incubated with 20 μl of Protein A Fast Flow Sepharose containing normal IgG for 2 h at 4 °C and centrifuged at 6,000 × g for 30 s and supernatants were collected and incubated with appropriate primary antibody (1:50 dilution or 1.2 μg/reaction) for 2 h at 4°C with rotating. Protein A Fast Flow Sepharose (20 μl) was added into the mixtures and incubated overnight. The lysates were centrifuged, after which the supernatants were collected as the unbound fraction. The pellets were washed and boiled in 40 μl of 2× SDS sample buffer containing β-mercaptoethanol for 5 min. The mixtures were centrifuged and the whole supernatants were loaded onto 10% SDS-PAGE gels for immunoblot analysis using standard methods (
      • Ing D.J.
      • Zang J.
      • Dzau V.J.
      • Webster K.A.
      • Bishopric N.H.
      ).
      Gel Filtration—Neonatal rat heart ventricles were homogenized in cell fractionation buffer, incubated on ice for 30 min, and then centrifuged for 15 min at 14,000 × g. Supernatants (cytosolic lysates) were collected and measured for protein concentration. Approximately 2.5 mg of cytosolic lysates (∼4 mg/ml) were incubated with or without cytochrome c (10 μm) and ATP (2 mm) for 30 min at 37 °C. Lysates were loaded into 75/16 Sepharose S300 HR gel filtration columns pre-equilibrated with cell fractionation buffer, and eluted by gravity at a flow rate of ∼0.3 ml/min. Fractions were collected at an interval of 7 min each, precipitated with trichloroacetic acid, and run on a 10% SDS-PAGE gel for immunoblot analysis.
      In Vitro Caspase Activity DeterminationIn vitro colorimetric assays of caspase-9 and caspase-3 activity were performed using a commercially available kit (Caspase-9 or Caspase-3 Colorimetric Assays, R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Briefly, cells were washed with ice-cold phosphate-buffered saline and harvested in lysis buffer. Lysates were mixed with chromophore-conjugated caspase-9- or caspase-3-specific substrates (LEHD-p-nitroaniline or DEVD-p-nitroaniline, respectively) in 96-well plates, incubated for 1 h at 37°C and assayed by measuring the emission wavelength for p-nitroaniline at 405 nm. Caspase activity for each sample was normalized to protein content. Lysates from cells treated with staurosporine (1 μm) for 8 h were used as a positive control.
      Statistical Analysis—Results were subjected to analysis of variance to determine significant inter-group differences, followed by Student's t test (InStat, GraphPad Inc., San Diego, CA). A p value of less than or equal to 0.05 was considered significant.

      RESULTS

      Dominant Negative JNK Blocks H2O2-induced JNK Activation—Consistent with earlier reports (
      • Laderoute K.R.
      • Webster K.A.
      ,
      • Clerk A.
      • Michael A.
      • Sugden P.H.
      ), H2O2 induced transient activation of Jun kinases 1 and 2 in a concentration- and time-dependent manner. A direct relationship between JNK activation and H2O2 concentration was observed up to 200 μm (Fig. 1A). Phosphorylation of both JNK-1 and JNK-2 was detectable at 15 min, maximal between 1 and 2 h, and back to baseline by 4 h (Fig. 1B). Infection of cardiomyocytes with a dominant negative JNK adenovirus almost eliminated JNK activation by H2O2 without affecting the activation level of extracellular-signal regulated kinase (ERK) or p38 (Fig. 1C and data not shown) and also blocked JNK activation by a maximally effective concentration of GSNO (Ref.
      • Andreka P.
      • Dougherty C.
      • Slepak T.I.
      • Webster K.A.
      • Bishopric N.H.
      and data not shown). A second inhibitory JNK mutant (JIA) (
      • Zheng C.
      • Xiang J.
      • Hunter T.
      • Lin A.
      )), a fusion of JNKK2 and the inactive JNK1 (APY) mutant, did not block H2O2-stimulated JNK phosphorylation in neonatal cardiomyocytes, but did block the phosphorylation of c-Jun (not shown), suggesting that the JIA mutant competes with endogenous JNK for occupancy of downstream targets. JNK activation induced by H2O2 could also be blocked by the anthrapyrazolone SP600125 (2 μm), a reversible ATP-competitive inhibitor of JNK with an in vivo IC50 of ∼5 μm and at least 10-fold in vivo selectivity for JNK compared with ERK or p38 (Fig. 1D) (
      • Krumenacker J.S.
      • Kots A.
      • Murad F.
      ,
      • Clerk A.
      • Kemp T.J.
      • Harrison J.G.
      • Mullen A.J.
      • Barton P.J.
      • Sugden P.H.
      ,
      • Bennett B.L.
      • Sasaki D.T.
      • Murray B.W.
      • O'Leary E.C.
      • Sakata S.T.
      • Xu W.
      • Leisten J.C.
      • Motiwala A.
      • Pierce S.
      • Satoh Y.
      • Bhagwat S.S.
      • Manning A.M.
      • Anderson D.W.
      ).
      Figure thumbnail gr1
      FIGURE 1Hydrogen peroxide induces Jun kinase activity in a dose- and time-dependent manner. A, concentration-dependent induction of JNK phosphorylation by H2O2. Cells were treated with varying concentrations of H2O2 for 1 h. Total cell lysates were immunoblotted with antibodies against phosphorylated JNK, total JNK, and actin. Anisomycin, 10 μg/ml (Ani), was used as a positive control. Graph shows the ratio of pJNK levels in treated cells to total JNK in simultaneous controls (n = 3). B, dose-dependent induction of JNK phosphorylation by H2O2. Cells were treated with 200 μm H2O2 at varying time points and lysates were visualized and analyzed as in A. C, dominant negative Jun kinase blocks JNK phosphorylation by H2O2. Cells were infected with adenovirus expressing GFP (empty vector) or dominant negative JNK (DNJ) for 48 h and treated with H2O2 (200 μm) for 1 h. Immunoblot analysis was performed as in A. D, chemical inhibition of H2O2-mediated JNK activation. Cells were incubated with SP600125 (SP, 2μm) a specific JNK inhibitor, for 30 min and then with H2O2 (200 μm) for 1 h. Immunoblot analysis was performed as in A.*, p < 0.05. d.u., density unit.
      Jun Kinase Delays Oxidant-induced Caspase-9 Activation—We have previously shown that nitric oxide donors induce time- and dose-dependent cardiac myocyte apoptosis through oxidant mechanisms, and that inhibition of Jun kinase accelerates the rate of GSNO-induced DNA fragmentation (
      • Andreka P.
      • Dougherty C.
      • Slepak T.I.
      • Webster K.A.
      • Bishopric N.H.
      ,
      • Ing D.J.
      • Zang J.
      • Dzau V.J.
      • Webster K.A.
      • Bishopric N.H.
      ). Consistent with these observations, we found that NO specifically activated the mitochondria-mediated cell death pathway. GSNO (1 mm) induced caspase-9 activation in a time-dependent manner (Fig. 2A) without affecting caspase-8 activity (Fig. 2B) or cleavage of its substrate, Bid (not shown). Inhibition of JNK by Ad-dnJNK accelerated the onset of LEHDase activity by 4–5 h, and increased LEHDase activity over GFP-infected cells at all time points between 0 and 3 h for GSNO (Fig. 2A) and 0 and 8 h for H2O2 (Fig. 2C). Ad-dnJNK accelerated a corresponding rise in DEVDase activity, indicating activation of the caspase-9 substrate caspase-3 (Fig. 2D), whereas also enhancing H2O2-mediated caspase-3 cleavage (Fig. 2, E and F), DNA internucleosomal cleavage (Fig. 2G), and apoptotic nuclear condensation (Fig. 2H). Similar results were observed in cells infected with adenovirus expressing the inactive JNK (Ad-JIA) in which JNK inhibition by Ad-JIA accelerated LEHDase and DEVDase activity induced by H2O2 (data not shown). These results show that oxidant-induced JNK activation delays activation of caspases-3 and -9, and subsequent apoptosis, by two different sources of oxidant stress. Because nitric oxide and H2O2 activate both overlapping and distinct intracellular signal pathways, this finding is consistent with JNK exerting cytoprotective effects at a common downstream effector of cell death.
      Figure thumbnail gr2
      FIGURE 2Jun kinase delays oxidant-induced caspase activation and DNA fragmentation. A and B, nitric oxide induces JNK-sensitive caspase-9, but not caspase-8 activity. Myocyte cultures were treated with GSNO (1 mm) for the indicated times. Total cell lysates were analyzed for caspase-9 (A) or caspase-8 (B) activity in vitro using a colorimetric assay. Cardiomyocytes treated with staurosporine (1μm Stau, gray circle) or Jurkat T cells treated for 24 h with TNF-α (20 ng/ml, open circle) were used as positive controls for activation of caspase-9 and -8, respectively. C and D, JNK delays H2O2-induced caspase-9 and -3 activation. Uninfected cells, or cells infected with Ad-GFP or Ad-DNJ, were treated with H2O2 (200 μm) for the indicated times and lysates analyzed for LEHDase (caspase-9) (C) or DEVDase (caspase-3) (D) activity in vitro as described in A. E and F, JNK inhibits caspase-3 cleavage. E, representative Western blot of Ad-GFP or Ad-DNJ-infected cells treated with H2O2 as in C, probed with an anti-caspase-3 antibody to monitor its cleavage from an inactive 32-kDa precursor (ProC3) to the active 20-kDa product (p20). B, BocD (pancaspase inhibitor); St, staurosporine (1 μm); C, control (no treatment). F, quantitation of three experiments in which essentially similar results were obtained. Data are represented as the relative increase in p20 band density at each time point normalized to the loading control actin. d.u., density unit. G, JNK inhibits oxidant-induced DNA fragmentation. Cells infected for 48 h with Ad-GFP or Ad-DNJ were treated with H2O2 as above and subjected to DNA fragmentation analysis by gel electrophoresis. H, JNK inhibits apoptic nuclear condensation. Cells treated as in panel G were stained with Hoechst 33342 and scored for nuclear condensation and fragmentation. At least 200 cells were counted per data point. Data represent at least three individual experiments. Statistical analysis was performed with analysis of variance followed by post hoc unpaired Student's t test. *, p < 0.05.
      NO-induced Cytochrome c Release Is Independent of JNK Activity—The selective activation of caspase-9 implicates the type II, mitochondria-mediated pathway in oxidant-mediated cardiomyocyte death. Under oxidant stress, mitochondria release cytochrome c, an essential cofactor for the formation of the apoptosome and subsequent activation of caspase-9 (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ,
      • Bossy-Wetzel E.
      • Newmeyer D.D.
      • Green D.R.
      ); JNK activation has been implicated in cytochrome c release from mouse embryo fibroblast mitochondria (
      • Tournier C.
      • Hess P.
      • Yang D.D.
      • Xu J.
      • Turner T.K.
      • Nimnual A.
      • Bar-Sagi D.
      • Jones S.N.
      • Flavell R.A.
      • Davis R.J.
      ), and from isolated cardiac mitochondria (
      • Aoki H.
      • Kang P.M.
      • Hampe J.
      • Yoshimura K.
      • Noma T.
      • Matzuzaki M.
      • Izumo S.
      ). To assess the effects of JNK activation on cytochrome c release in intact cardiac myocytes, we monitored cytosolic levels of cytochrome c release in cells treated with GSNO in the presence and absence of dnJNK. In uninfected cardiac myocytes, GSNO (1 mm) induced the release of cytochrome c into the cytosol within 10 min of treatment; by 5 h after treatment, cytosolic cytochrome c levels approached those of control cells (Fig. 3). The disappearance of cytochrome c from cytosolic fractions at these time points most likely reflects loss of apoptotic cells from the culture dish; however, it is also possible that cytochrome c is taken back up by the mitochondria or otherwise eliminated in surviving cells, as proposed elsewhere (
      • Von Ahsen O.
      • Waterhouse N.J.
      • Kuwana T.
      • Newmeyer D.D.
      • Green D.R.
      ). Identical amounts and timing of cytochrome c release were seen in cells infected either with a blank virus (GFP) or Ad-dnJNK. In contrast, overexpression of Bcl-XL delayed cytochrome c release by 12 h (Fig. 3). These results show that active JNK is not required for initiation of the mitochondrial apoptotic pathway in intact cardiac myocytes, and imply that JNK modulates caspase-9 activation through a post-mitochondrial mechanism.
      Figure thumbnail gr3
      FIGURE 3Jun kinase activity has no effect on the rate of NO-induced cytochrome c release. Cardiomyocytes infected with the indicated viruses (Ad-β-gal (β-Gal), Ad-dnJNK (DNJ), or Ad-Bcl-XL (Bcl-XL)) were treated with GSNO (1 mm) for the indicated times. Samples of cytosolic and mitochondrial fractions were immunoblotted for cytochrome c (Cyt c) and cytochrome c oxidase subunit IV (COX). The graph summarizes densitometric results from three independent experiments, with cytosolic cytochrome c density expressed as a percentage of maximum (density at 2 h in uninfected cells); representative immunoblots are shown. Cytosol, cytosolic fraction; mitochondria, mitochondrial fraction. d.u., density unit.
      Jun Kinase Prevents Caspase Activation in a Cell-free System—Human caspase-9 contains phosphorylation sites for ERK and Akt (
      • Allan L.A.
      • Morrice N.
      • Brady S.
      • Magee G.
      • Pathak S.
      • Clarke P.R.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), which may also be activated during oxidant stress. To avoid these potentially confounding effects, we examined the effects of JNK activity on caspase-9 and caspase-3 processing in the absence of oxidant stress, using a previously characterized cell- and organelle-free system (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ). Cytosolic extracts of cardiac myocytes and HEK293 cells expressing Ad-GFP or Ad-dnJNK were incubated with or without cytochrome c and ATP, and cleavage of caspase-3 and -9 was monitored by Western analysis. After 3 h of incubation with cytochrome c and ATP, the p37 and p20 cleavage products of caspases-9 and -3, respectively, were significantly more abundant in cardiac myocyte cytosol lacking basal JNK activity (Figs. 4, A–C). However, in HEK293 cytosolic lysates, JNK inhibition had no effect on the processing of either caspase-3 or the caspase-3 substrate inhibitor of caspase-activated DNase (Fig. 4, D–F). This finding confirms that active JNK interferes with cytosolic caspase-9 activation at a step distal to the mitochondrial release of cytochrome c. Furthermore, this effect appears to be cell type-restricted.
      Figure thumbnail gr4
      FIGURE 4Jun kinase inhibits caspase activation in a cell-free system. A–C, JNK inhibits in vitro processing of caspase-9 and -3 in cardiac myocytes. Cytosolic lysates of cardiomyocytes (A–C) infected with adenoviruses expressing GFP, dnJNK (DNJ), or a dominant negative caspase-9 (DNC9) were examined directly, or incubated for 3 h at 37 °C with or without cytochrome c (10 μm) and ATP (1 mm), and samples (50 μg each) immunoblotted as shown. A, Western blot analysis of cardiomyocyte cytosolic caspase-9 (p37, upper) and caspase-3 (p20, lower). B, quantitation of caspase-9 cleavage product p37, normalized to actin (n = 3). C, quantitation of caspase-3 cleavage product, normalized to actin (n = 3). D–F, JNK does not inhibit post-mitochondrial caspase-3 processing in HEK293 cells. D, Western blot analysis of caspase-3 and inhibitor of caspase-activated DNase cleavage in the cytosol of HEK293 cells, before incubation, or after 30 min incubation ± cytochrome c and ATP as in A–C. Data represent at least three individual experiments. E, densitometry measurements of caspase-3 p20 cleavage product. F, inhibitor of caspase-activated DNase cleavage densitometry. Band intensities were normalized as in B and C.*, p < 0.05. ProCasp9, procaspase 9. Casp3 p20, caspase 3 cleavage product. d.u., density unit.
      JNK Activation Retards Apoptosome Formation—The critical step in activation of procaspase-9 is not proteolytic processing but dimerization, potentiated by enforced aggregation within the apoptosome (
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Zou H.
      • Li Y.
      • Liu X.
      • Wang X.
      ,
      • Cain K.
      • Bratton S.B.
      • Langlais C.
      • Walker G.
      • Brown D.G.
      • Sun X.M.
      • Cohen G.M.
      ,
      • Rodriguez J.
      • Lazebnik Y.
      ,
      • Renatus M.
      • Stennicke H.R.
      • Scott F.L.
      • Liddington R.C.
      • Salvesen G.S.
      ,
      • Boatright K.M.
      • Renatus M.
      • Scott F.L.
      • Sperandio S.
      • Shin H.
      • Pedersen I.M.
      • Ricci J.E.
      • Edris W.A.
      • Sutherlin D.P.
      • Green D.R.
      • Salvesen G.S.
      ,
      • Pop C.
      • Timmer J.
      • Sperandio S.
      • Salvesen G.S.
      ). Accordingly, we assessed the effect of JNK inhibition on the association of Apaf-1 and caspase-9 in cells under oxidant stress. Complex formation between caspase-9 and Apaf-1 was first detectable 3 h after GSNO stress in uninfected cells or cells expressing a blank vector (Fig. 5). In JNK-inhibited cells, caspase-9-Apaf-1 interaction appeared at least 2 h earlier, i.e. within 1 h after GSNO treatment. This difference corresponds closely to the 2–3-h acceleration in caspase-9 induction observed with JNK inhibition (compare Fig. 2, A and B), and suggests that activation of JNK by oxidant stress may create a several-hour delay in the formation of an active apoptosome.
      Figure thumbnail gr5
      FIGURE 5JNK inhibition accelerates association of Apaf-1 and caspase-9 after oxidant stress. The interaction of caspase-9 and Apaf-1 was monitored in control cardiomyocytes, or cardiomyocytes infected with Ad-β-galactosidase (β-Gal) or Ad-dnJNK (DNJ) (see “Experimental Procedures”). Total cell lysates were prepared from cardiomyocytes after treatment with GSNO (1 mm) for the indicated times. A portion of the lysates was immunoprecipitated (IP) with antibody against caspase-9, and immunoblotted for Apaf-1. The remaining total cell lysates were immunoblotted for total caspase-9 and Apaf-1. Upper panel, representative immunoblots for caspase-9 immunoprecipitates and total cell lysates at the indicated times after GSNO treatment. Lower panel, densitometric analysis of caspase-9-complexed Apaf-1 (n = 3) normalized to total Apaf-1 (n = 3, **, p < 0.05). d.u., density unit.
      JNK Activation and Cytochrome c Promote JNK-Apaf-1 Interaction—To determine how JNK might affect caspase-9 interaction with Apaf-1, we asked whether JNK itself could bind to apoptosome components. In cells treated with either GSNO or H2O2, JNK and caspase-9 did not interact (not shown); however, JNK and Apaf-1 were readily and reciprocally co-immunoprecipitated (Fig. 6A). Oxidant stress strongly potentiated this interaction. Apaf-1-JNK complexes were significantly increased in cytosol from cells treated with GSNO (3.2 ± 0.82-fold versus control, p < 0.05) or H2O2 (5.7 ± 1.21-fold, p < 0.05) compared with cytosol from untreated cells (Fig. 6, B, lanes 1 and 2, C, lanes 1 and 2; and data not shown). This increase was at least partly due to oxidant-stimulated JNK activity, because JNK interaction with Apaf-1 was markedly reduced either by co-expression of dnJNK (Fig. 6B, lanes 3 and 4) or treatment with the JNK inhibitor SP600125 over a ×100 range of concentrations (Fig. 6C, lanes 2 and 3). Consistent with this, the dnJNK mutant protein itself failed to associate with endogenous Apaf-1 (Fig. 6B and data not shown). In parallel experiments, adenovirus-mediated overexpression of a wild type JNK1 linked to an inactive JNKK2(KM) (
      • Zheng C.
      • Xiang J.
      • Hunter T.
      • Lin A.
      ) had no effect on endogenous JNK activity, and also had no effect on the interaction between endogenous JNK and Apaf-1 (data not shown). Thus, abrogation of the endogenous Apaf1-JNK complex by dnJNK1 was likely attributable to the ability of dnJNK to prevent JNK activation and not to JNK overexpression generally.
      Figure thumbnail gr6
      FIGURE 6Cytochrome c potentiates a stable interaction between activated Jun kinase and Apaf-1. A, JNK-Apaf-1 interaction in GSNO-treated cardiomyocytes. Total cell lysates were prepared from GSNO-treated cardiomyocytes, immunoprecipitated (IP) with antibody against either JNK (upper panel) or Apaf-1 (lower panel) and subjected to Western analysis for Apaf-1 or JNK, respectively. The corresponding positions of immunoprecipitated, immunoblotted (IB) Apaf-1 (upper panel, first lane) and JNK (lower panel, fourth lane) are shown to confirm the identity of the co-precipitated proteins. No JNK or Apaf-1-reactive bands were detected when immunoprecipitation was carried out with an unrelated antibody (MEF-2, fifth lane, both panels) or IgG (sixth lane, both panels). Only the p46JNK isoform was detectable in Apaf-1 immunoprecipitates. B, dominant negative JNK inhibits Apaf-1-JNK interaction. Cardiomyocytes expressing DNJ or GFP were treated with H2O2 (200 μm) or vehicle for 1 h. Cytosolic lysates were immunoprecipitated with an anti-JNK antibody and then immunoblotted with antibodies against Apaf-1 and JNK (top two blots), or immunoblotted directly (Total Cell Lysate). Upper panel, representative immunoblots. The dominant negative JNK mutant protein (DNJ) can be seen in both JNK immunoprecipitates and total cell lysates (second and fourth blots, third and fourth lanes), but evidently does not interact with Apaf-1 because it reduces the total amount of co-immunoprecipitated Apaf-1 (top blot, lanes 3 and 4). Lower panel, graph summarizes data from three separate experiments, in which the Apaf-1 band density was normalized to co-precipitated endogenous JNK. -Fold induction was calculated by dividing the normalized density units of treated samples by those of untreated samples (T/C). Results are presented as mean ± S.E. C, SP600125 inhibits Apaf-1-JNK interaction. Cardiomyocytes were exposed to H2O2 in the presence and absence of 0.2–20 μm JNK inhibitor SP600125 as shown, and cytosolic lysates were analyzed for Apaf-1 co-immunoprecipitating with JNK as in B. Top, representative immunoblots for Apaf-1 and JNK. IgG, control immunoprecipitation with IgG. Unb, unbound fraction of immunoprecipitate. Asterisks indicate nonspecific bands. Center, immunoblots of total cell lysates from control cells and cells treated with H2O2 in the presence and absence of 1μm SP600125. Bottom, graph summarizing three independent experiments, with normalization as in B.*, p < 0.05. n.d.u., normalized density unit. D, cytochrome c potentiates Apaf-1-JNK interaction. Top, cytosolic lysates (100μg) of untreated cells infected with either Ad-GFP (GFP) or Ad-dnJNK (DNJ) were incubated with or without cytochrome c/ATP for 30 min at 37 °C, immunoprecipitated with an anti-JNK antibody, and immunoblotted for Apaf-1 (upper gel) or JNK (lower gel). Bottom, quantitation of three experiments was performed as in B.*, p < 0.05. d.u., density unit.
      The oxidant conditions leading to cellular JNK activation also induce the release of mitochondrial cytochrome c, which promotes the oligomerization of Apaf-1, as well as a number of other signaling events. To study the formation of the Apaf-1-JNK complex in the absence of oxidants, we again utilized an in vitro system in which cytochrome c and ATP or their vehicle were added directly to cytosol purified from unstressed cells. Cytosolic JNK-Apaf-1 interaction increased significantly after addition of cytochrome c (7.5 ± 1.7-fold versus vehicle, p < 0.05; Fig. 6D, lanes 1 and 2). As with intact cells, inhibition of JNK reduced basal JNK-Apaf-1 interaction (Fig. 6D, lanes 1 and 4) and also completely prevented the cytochrome c-mediated increase in this complex (0.8 ± 0.2-fold versus vehicle) (Fig. 6D, lanes 4 and 5). Thus, both cytochrome c and JNK activity regulate the interaction between JNK and Apaf-1, independent of other oxidant-mediated events.
      Active JNK Associates with Oligomerized Apaf-1 and Cytochrome c—Cytochrome c could stimulate Apaf-1-JNK binding by promoting a conformation of Apaf-1 with which JNK preferentially associates. To explore this possibility, we assessed the size and composition of the cytochrome c-induced JNK-Apaf-1 complex using size-exclusion chromatography. Cell-free, mitochondria-depleted cytosolic extracts were prepared from normal rat ventricular muscle and incubated with or without exogenous cytochrome c and ATP for 30 min at 37 °C. Samples were fractionated by size exclusion chromatography and subjected to Western analysis for Apaf-1, JNK1/2, X-linked inhibitor of apoptosis (XIAP), and cytochrome c. Consistent with previous studies, the majority of Apaf-1 eluted as part of two large multimeric complexes, estimated at ≥1.4 mDa and 440–700 kDa based on the elution of size markers (complexes I and II, respectively, Fig. 7A) (
      • Zou H.
      • Li Y.
      • Liu X.
      • Wang X.
      ,
      • Cain K.
      • Bratton S.B.
      • Langlais C.
      • Walker G.
      • Brown D.G.
      • Sun X.M.
      • Cohen G.M.
      ,
      • Rodriguez J.
      • Lazebnik Y.
      ,
      • Saleh A.
      • Srinivasula S.M.
      • Acharya S.
      • Fishel R.
      • Alnemri E.S.
      ,
      • Beem E.
      • Holliday L.S.
      • Segal M.S.
      ). Almost all detectable p46JNK, and a small amount of p54JNK, co-eluted with Apaf-1 as part of the larger of these complexes, together with cytochrome c and some p37 caspase-9 (Fig. 7A, complex I). The second, more rapidly eluting complex contained oligomeric Apaf-1 and XIAP and was consistent in size with the active apoptosome (
      • Cain K.
      • Bratton S.B.
      • Langlais C.
      • Walker G.
      • Brown D.G.
      • Sun X.M.
      • Cohen G.M.
      ). Neither of these complexes was detected in the absence of added cytochrome c/ATP (not shown). Processed caspase-9 (p37) was also detectable in monomeric fractions (Fig. 7A, III). No caspase-3 or DEVDase activity was detected in any fraction (Fig. 7A and data not shown), possibly reflecting the association of XIAP with the 440–700 kDa complex (Fig. 7A, complex II), as previously reported for non-apoptotic cells (
      • Twiddy D.
      • Brown D.G.
      • Adrain C.
      • Jukes R.
      • Martin S.J.
      • Cohen G.M.
      • MacFarlane M.
      • Cain K.
      ).
      Figure thumbnail gr7
      FIGURE 7JNK delays formation of the active apoptosome. A, Apaf-1 and JNK associate in a ∼1.4 mDa complex. Cytosolic lysates (2.5 mg) were obtained by hypotonic lysis of neonatal rat ventricular myocardium, programmed with cytochrome c/ATP for 30 min at 37 °C, and separated on a sizing column as described under “Experimental Procedures.” Proteins in each size fraction were then separated on 10% SDS-PAGE gels and immunoblotted with antibodies against Apaf-1, JNK1/2, cytochrome c, caspase-9, caspase-3, and XIAP. Positions of major Apaf-1-containing fractions (I and II) and XIAP-containing fraction (II) as well as fractions containing largely monomeric proteins (III) are indicated by brackets. Fraction numbers are shown; Apaf-1 and JNK co-elute with a peak at fraction 30. Arrows represent peak of elution of molecular standards: black, blue dextran 2000 (2 mDa); light gray, thyroglobulin (669 kDa); dark gray, ferritin (440 kDa); white, aldolase (158 kDa); and medium gray, bovine serum albumin (67 kDa). For this column, the end of the void volume precedes elution of blue dextran 2000. Upper, representative immunoblots of sequential fractions. Lower, summary of three independent experiments. Densitometry values were obtained using Adobe Photoshop 7.0 and are plotted as percentages of the maximum band density for each blot. B, dimerization of procaspase-9 in JNK-inhibited cells. Cytosolic lysates were prepared from cultured unstimulated cardiac myocytes infected with empty adenovirus vector expressing GFP only (Blank) or Ad-dnJNK (DNJ) were programmed with cytochrome c/ATP for 15 min at 37 °C and separated on sizing columns as in A. Fractions were analyzed by immunoblotting for the indicated proteins. Density units were measured as above and normalized to co-migrating Ponceau-stained proteins on the membrane. Representative immunoblots are shown. CE, summaries of a minimum of three experiments showing comparative elution profiles of Apaf-1 (C), caspase-9 (D), and caspase-3 (E). n.d.u., normalized density unit.
      To determine how JNK activity affected the distribution of these protein complexes, cytosolic lysates were prepared from unstimulated cardiac myocytes expressing Ad-GFP (GFP) or Ad-dnJNK (DNJ), primed briefly (15 min) with cytochrome c and ATP, and subjected to size fractionation as above. At this early time point, no caspase-3 cleavage or processing activity was detectable in either lysate, and procaspase-3 was exclusively present in low Mr fractions (Fig. 7E and data not shown). Similar to our findings in myocardial cytosol, exogenous cytochrome c eluted in low Mr fractions, possibly complexed with other proteins, and also in oligomeric complexes with Apaf-1 (compare Fig. 7, A and B). Expression of the JNK inhibitor increased the amount of Apaf-1 present in the 440–700-kDa complex by 4–5-fold compared with GFP alone (Fig. 7, B and C, fractions 32–38), suggesting an increase in oligomeric Apaf-1. In addition, a band consistent with the 70–80-kDa caspase-9 dimer was associated with the smaller Apaf-1 complex only in JNK-inhibited cells (Fig. 7, B and D, complex II). These findings are consistent with a mechanism by which JNK retards the activation of caspase-9 by preventing its association with Apaf-1.

      DISCUSSION

      We show here that upon activation by oxidant stress, cardiac myocyte JNK forms a stable complex with oligomerized Apaf-1. Association between JNK-Apaf-1 appears to be independent of the upstream events leading to JNK activation. Nitric oxide activates JNK through the cyclic GMP pathway (
      • Ing D.J.
      • Zang J.
      • Dzau V.J.
      • Webster K.A.
      • Bishopric N.H.
      ), whereas reactive oxygen species such as H2O2 induce JNK activation through the MAPKKK ASK1 (
      • Saitoh M.
      • Nishitoh H.
      • Fujii M.
      • Takeda K.
      • Tobiume K.
      • Sawada Y.
      • Kawabata M.
      • Miyazono K.
      • Ichijo H.
      ,
      • Liu H.
      • Nishitoh H.
      • Ichijo H.
      • Kyriakis J.M.
      ); nonetheless, both signals induced the formation of this complex.
      Although the precise relationship between the JNK-Apaf-1 complex and the canonical apoptosome remains to be elucidated, we also provide evidence that formation of this complex is associated with a delay in assembly of the active apoptosome and in activation of caspase-9. The timing and magnitude of these effects coincide with the peak in JNK activity. Both JNK activation and caspase inhibition are time-limited; however, it is likely that the downstream effects are more sustained. Because caspase-9 is catalytic, changes in its activity are magnified significantly at the level of caspase-3 activation, and more so at the level of terminal substrates such as DNA.
      JNK has been shown to modulate mitochondria-dependent apoptosis by phosphorylating members of the Bcl-2 family, which control the release of cytochrome c into the cytosol during apoptosis. Most of these modifications are pro-apoptotic (
      • Donovan N.
      • Becker E.B.E.
      • Konishi Y.
      • Bonni A.
      ,
      • Kharbanda S.
      • Saxena S.
      • Yoshida K.
      • Pandey P.
      • Kaneki M.
      • Wang Q.
      • Cheng K.
      • Chen Y.N.
      • Campbell A.
      • Sudha T.
      • Yuan Z.M.
      • Narula J.
      • Weichselbaum R.
      • Nalin C.
      • Kufe D.
      ,
      • Inoshita S.
      • Takeda K.
      • Hatai T.
      • Terada Y.
      • Sano M.
      • Hata J.
      • Umezawa A.
      • Ichijo H.
      ,
      • Yamamoto K.
      • Ichijo H.
      • Korsmeyer S.J.
      ,
      • Deng X.
      • Xiao L.
      • Lang W.
      • Gao F.
      • Ruvolo P.
      • May Jr., W.S.
      ,
      • Lei K.
      • Davis R.J.
      ). However, in our system JNK inhibition did not affect the timing of cytochrome c release, arguing against an important effect of JNK on mitochondrial permeability. JNK has been reported to block the pro-apoptotic up-regulation of TNF-α (
      • Minamino T.
      • Yujiri T.
      • Papst P.J.
      • Chan E.D.
      • Johnson G.L.
      • Terada N.
      ), and to potentiate the cytoprotective effects of XIAP in MCF and HEK293 cells (
      • Sanna M.G.
      • Duckett C.S.
      • Richter B.W.
      • Thompson C.B.
      • Ulevitch R.J.
      ). Because apoptosis in our system occurs independently of TNF-α, caspase-8, and Bid cleavage, neither of these mechanisms explains JNK cytoprotection in our system (Fig. 2A and Ref.
      • Ing D.J.
      • Zang J.
      • Dzau V.J.
      • Webster K.A.
      • Bishopric N.H.
      ). JNK can also act to prevent cell death by transcriptional induction of survival genes, including JunD and cIAP (
      • Lamb J.A.
      • Ventura J.J.
      • Hess P.
      • Flavell R.A.
      • Davis R.J.
      ); whether transcriptional effects modulate formation of the JNK-Apaf-1 complex remains to be determined. Cell type- or developmental stage-specific factors may also be involved in JNK cytoprotection, because inhibition of JNK did not promote, and may actually have reduced, caspase-3 processing and activity in HEK293 cells (see Fig. 4, D–F). JNK effects on cell fate are known to be context-dependent (
      • Kuan C.Y.
      • Yang D.D.
      • Samanta Roy D.R.
      • Davis R.J.
      • Rakic P.
      • Flavell R.A.
      ,
      • Sabapathy K.
      • Hu Y.
      • Kallunki T.
      • Schreiber M.
      • David J.P.
      • Jochum W.
      • Wagner E.F.
      • Karin M.
      ,
      • Dong C.
      • Yang D.D.
      • Tournier C.
      • Whitmarsh A.J.
      • Xu J.
      • Davis R.J.
      • Flavell R.A.
      ,
      • David J.P.
      • Sabapathy K.
      • Hoffmann O.
      • Idarraga M.H.
      • Wagner E.F.
      ,
      • Leppa S.
      • Bohmann D.
      ,
      • Brecht S.
      • Kirchhof R.
      • Chromik A.
      • Willesen M.
      • Nicolaus T.
      • Raivich G.
      • Wessig J.
      • Waetzig V.
      • Goetz M.
      • Claussen M.
      • Pearse D.
      • Kuan C.Y.
      • Vaudano E.
      • Behrens A.
      • Wagner E.
      • Flavell R.A.
      • Davis R.J.
      • Herdegen T.
      ,
      • Nishitai G.
      • Shimizu N.
      • Negishi T.
      • Kishimoto H.
      • Nakagawa K.
      • Kitagawa D.
      • Watanabe T.
      • Momose H.
      • Ohata S.
      • Tanemura S.
      • Asaka S.
      • Kubota J.
      • Saito R.
      • Yoshida H.
      • Mak T.W.
      • Wada T.
      • Penninger J.M.
      • Azuma N.
      • Nishina H.
      • Katada T.
      ).
      Sizing chromatography confirmed the association of JNK1/2 and Apaf-1 in a complex with an effective molecular mass of 1.4–2.0 mDa, similar to the previously described 1.4-mDa Apaf-1-containing complex in Jurkat T and monocyte cell lysates (
      • Zou H.
      • Li Y.
      • Liu X.
      • Wang X.
      ,
      • Saleh A.
      • Srinivasula S.M.
      • Acharya S.
      • Fishel R.
      • Alnemri E.S.
      ). In those cells, two Apaf-1-containing complexes of ∼700 kDa and 1.4 mDa could associate with caspase-9, but most caspase-processing activity was found in the 700-kDa complex (
      • Cain K.
      • Bratton S.B.
      • Langlais C.
      • Walker G.
      • Brown D.G.
      • Sun X.M.
      • Cohen G.M.
      ,
      • Saleh A.
      • Srinivasula S.M.
      • Acharya S.
      • Fishel R.
      • Alnemri E.S.
      ,
      • Beem E.
      • Holliday L.S.
      • Segal M.S.
      ). Matrix-assisted laser desorption ionization time-of-flight spectrometry of the 700-kDa complex in THP.1 monocytes identified Apaf-1 and caspase-9 as the major components, with XIAP also present in non-apoptotic cells (
      • Twiddy D.
      • Brown D.G.
      • Adrain C.
      • Jukes R.
      • Martin S.J.
      • Cohen G.M.
      • MacFarlane M.
      • Cain K.
      ). Likewise, we found no JNK in the 700-kDa complex. It has been reported that recombinant Apaf-1 and caspase-9 spontaneously form a >1.3 mDa complex in the presence of cytochrome c and ATP with a stoichiometry of 1:1 (
      • Zou H.
      • Li Y.
      • Liu X.
      • Wang X.
      ) and a heptameric hub-and-spoke structure (
      • Acehan D.
      • Jiang X.
      • Morgan D.G.
      • Heuser J.E.
      • Wang X.
      • Akey C.W.
      ). Because of limited resolution of the gel filtration columns at high molecular sizes, these studies, like the present one, have not established a precise molecular mass for the larger (1.4–2.0 mDa) complex, and therefore do not exclude the presence of JNK or other proteins.
      Our size fractionation studies further demonstrate that a 46-kDa protein is the major JNK species in the 1.4-mDa complex. In fact, almost all of the cytosolic 46-kDa JNK is found in that complex, whereas most of the 54-kDa JNK is found in monomeric fractions. Both p46 and p54 isoforms can arise from alternative splicing of each of the primary JNK transcripts encoded by the JNK-1, -2, and -3 genes; however, JNK-1 appears predominantly as the p46 splice variant, whereas JNK-2 and -3 are principally expressed as the p54 variant (
      • Liu J.
      • Minemoto Y.
      • Lin A.
      ,
      • Dreskin S.C.
      • Thomas G.W.
      • Dale S.N.
      • Heasley L.E.
      ). Apaf-1 thus appears to associate preferentially with JNK-1. Although JNK-1 and JNK-2 are functionally redundant to some extent, especially during development (
      • Kuan C.Y.
      • Yang D.D.
      • Samanta Roy D.R.
      • Davis R.J.
      • Rakic P.
      • Flavell R.A.
      ,
      • Sabapathy K.
      • Jochum W.
      • Hochedlinger K.
      • Chang L.
      • Karin M.
      • Wagner E.F.
      ), isoform-specific functions for these proteins have been identified in various tissues and cell types (
      • Sabapathy K.
      • Hu Y.
      • Kallunki T.
      • Schreiber M.
      • David J.P.
      • Jochum W.
      • Wagner E.F.
      • Karin M.
      ,
      • David J.P.
      • Sabapathy K.
      • Hoffmann O.
      • Idarraga M.H.
      • Wagner E.F.
      ). JNK-1 has both pro- and anti-apoptotic actions that depend on context, cell type, duration of activation, and co-activation of other signal transduction molecules (
      • Hochedlinger K.
      • Wagner E.F.
      • Sabapathy K.
      ). JNK-1 is the principal JNK species activated by TNF-α, UV exposure, and anisomycin (
      • Liu J.
      • Minemoto Y.
      • Lin A.
      ) and its activation is associated with obesity and onset of diabetes (
      • Hirosumi J.
      • Tuncman G.
      • Chang L.
      • Gorgun C.Z.
      • Uysal K.T.
      • Maeda K.
      • Karin M.
      • Hotamisligil G.S.
      ). JNK-1 exhibits both pro-survival and pro-death effects in the brain, where it is the most abundant phospho-JNK species (
      • Brecht S.
      • Kirchhof R.
      • Chromik A.
      • Willesen M.
      • Nicolaus T.
      • Raivich G.
      • Wessig J.
      • Waetzig V.
      • Goetz M.
      • Claussen M.
      • Pearse D.
      • Kuan C.Y.
      • Vaudano E.
      • Behrens A.
      • Wagner E.
      • Flavell R.A.
      • Davis R.J.
      • Herdegen T.
      ). In glucose-deficient cardiac myocytes, JNK-1 but not JNK-2 mediated oxidative stress-induced apoptosis (
      • Hreniuk D.
      • Garay M.
      • Gaarde W.
      • Monia B.P.
      • McKay R.A.
      • Cioffi C.L.
      ). On the other hand, suggesting a pro-survival for JNK-1 in the heart, mice with deletion of JNK-1, but not JNK-2, were shown to have impaired adaptation to pressure overload (
      • Tachibana H.
      • Perrino C.
      • Takaoka H.
      • Davis R.J.
      • Naga Prasad S.V.
      • Rockman H.A.
      ).
      These findings suggest that JNK retards the processing and enzymatic activation of caspase-9 in vivo by direct effects at the apoptosome. However, whether JNK has a direct substrate within the apoptosome remains to be determined. Although we find that JNK activity is crucial to induce the interaction with Apaf-1, it may be dispensable thereafter, because none of the JNK within the 1.4-mDa complex appears to be phosphorylated (not shown). Moreover, we have no evidence that JNK is able to phosphorylate Apaf-1; although preliminary evidence indicates that cardiac myocyte Apaf-1 is heavily serine-phosphorylated, the abundance of phospho-Apaf-1 is not directly related to JNK activity (data not shown). Sequence analysis of Apaf-1 reveals several consensus docking sites for JNK, and it is possible that the JNK-Apaf-1 interaction provides a scaffold for the recruitment of JNK-associated proteins such as MKK7 and/or MAPK-associated phosphatases that modulate phosphorylation of caspase-9, Apaf-1, or JNK itself. Full identification of the components of the 1.4–2.0 mDa Apaf-1 complex may shed light on these questions.
      Our results also show that in intact, non-stressed myocardium, oligomerized Apaf-1 is extensively bound to XIAP. XIAP provides a major line of defense against accidental or transient cytochrome c release, because it strongly inhibits apoptosome-dependent caspase-9 activation (
      • Bratton S.B.
      • Lewis J.
      • Butterworth M.
      • Duckett C.S.
      • Cohen G.M.
      ,
      • Bratton S.B.
      • Walker G.
      • Srinivasula S.M.
      • Sun X.M.
      • Butterworth M.
      • Alnemri E.S.
      • Cohen G.M.
      ). Some cytoprotective effects of XIAP have been linked to JNK-1 activation via TAK1 (
      • Sanna M.G.
      • da Silva Correia J.
      • Luo Y.
      • Chuang B.
      • Paulson L.M.
      • Nguyen B.
      • Deveraux Q.L.
      • Ulevitch R.J.
      ). Other factors that inhibit apoptosome-dependent caspase processing include heat-shock protein 70 (HSP70) and 90 (HSP90), protein kinase A, and taurine, a β-amino acid (
      • Beere H.M.
      • Wolf B.B.
      • Cain K.
      • Mosser D.D.
      • Mahboubi A.
      • Kuwana T.
      • Tailor P.
      • Morimoto R.I.
      • Cohen G.M.
      • Green D.R.
      ,
      • Saleh A.
      • Srinivasula S.M.
      • Balkir L.
      • Robbins P.D.
      • Alnemri E.S.
      ,
      • Pandey P.
      • Saleh A.
      • Nakazawa A.
      • Kumar S.
      • Srinivasula S.M.
      • Kumar V.
      • Weichselbaum R.
      • Nalin C.
      • Alnemri E.S.
      • Kufe D.
      • Kharbanda S.
      ,
      • Martin M.C.
      • Allan L.A.
      • Lickrish M.
      • Sampson C.
      • Morrice N.
      • Clarke P.R.
      ,
      • Takatani T.
      • Takahashi K.
      • Uozumi Y.
      • Shikata E.
      • Yamamoto Y.
      • Ito T.
      • Matsuda T.
      • Schaffer S.W.
      • Fujio Y.
      • Azuma J.
      ). HSP90 appears to prevent Apaf-1 oligomerization and interaction with cytochrome c (
      • Pandey P.
      • Saleh A.
      • Nakazawa A.
      • Kumar S.
      • Srinivasula S.M.
      • Kumar V.
      • Weichselbaum R.
      • Nalin C.
      • Alnemri E.S.
      • Kufe D.
      • Kharbanda S.
      ). HSP70 has been reported to bind to Apaf-1 and prevent recruitment of caspase-9 to the apoptosome, without affecting Apaf-1 aggregation (
      • Beere H.M.
      • Wolf B.B.
      • Cain K.
      • Mosser D.D.
      • Mahboubi A.
      • Kuwana T.
      • Tailor P.
      • Morimoto R.I.
      • Cohen G.M.
      • Green D.R.
      ). Rather than competing directly with caspase-9 for binding to Apaf-1, Hsp70 may stabilize an inactive Apaf-1 conformation. JNK is required for activation of the HSP70 promoter during heat stress (
      • Park J.
      • Liu A.Y.
      ), and it is possible that transcriptional induction of HSP70 could contribute to delayed cytoprotective effects of JNK activation.
      JNK activity in cardiac myocytes is dynamic, and sensitive both to cellular redox potential and contractile activity. Transient increases in oxidant stress that occur during periods of increased O2 demand (such as increased contractile activity), or reduced O2 supply are potent stimuli for JNK activation (
      • Laderoute K.R.
      • Webster K.A.
      ). Normal contractile activity and cyclic stretch of myocytes generate significant amounts of reactive oxygen species and probably account for relatively high basal JNK activity in cardiac myocytes (
      • Pinsky D.J.
      • Patton S.
      • Mesaros S.
      • Brovkovych V.
      • Kubaszewski E.
      • Grunfeld S.
      • Malinski T.
      ,
      • Pimentel D.R.
      • Amin J.K.
      • Xiao L.
      • Miller T.
      • Viereck J.
      • Oliver-Krasinski J.
      • Baliga R.
      • Wang J.
      • Siwik D.A.
      • Singh K.
      • Pagano P.
      • Colucci W.S.
      • Sawyer D.B.
      ) and for the observation that JNK is modulated in vivo in direct relation to cardiac work (
      • Boluyt M.O.
      • Loyd A.M.
      • Roth M.H.
      • Randall M.J.
      • Song E.Y.
      ,
      • Flesch M.
      • Margulies K.B.
      • Mochmann H.C.
      • Engel D.
      • Sivasubramanian N.
      • Mann D.L.
      ,
      • Communal C.
      • Colucci W.S.
      • Remondino A.
      • Sawyer D.B.
      • Port J.D.
      • Wichman S.E.
      • Bristow M.R.
      • Singh K.
      ). The extent of physiologic activation of JNK in the myocardium argues against a simple role in cell death signaling, a view supported by an elegant in vivo study by Kaiser et al. (
      • Kaiser R.A.
      • Liang Q.
      • Bueno O.F.
      • Huang Y.
      • Lackey T.
      • Klevitsky R.
      • Hewett T.E.
      • Molkentin J.D.
      ). We propose that JNK activation is a physiological adaptation that acts as a buffer against the frequent (but usually transient) bursts of oxidant stress to which the myocardium is exposed. Given that some of the initial steps in oxidant-stimulated, mitochondria-dependent apoptosis may be reversible, including cytochrome c release (
      • Seye C.I.
      • Knaapen M.W.
      • Daret D.
      • Desgranges C.
      • Herman A.G.
      • Kockx M.M.
      • Bult H.
      ,
      • Mootha V.K.
      • Wei M.C.
      • Buttle K.F.
      • Scorrano L.
      • Panoutsakopoulou V.
      • Mannella C.A.
      • Korsmeyer S.J.
      ), the ability of coupled JNK activation to retard the post-mitochondrial phase of apoptosis may be critical to its ability to limit myocyte loss under conditions of increased workload or oxygen deficit.

      Acknowledgments

      We are grateful to Jian Qin Wei, Lijing You, and Grazia Spiga for insightful critical comments during the performance of these studies and to Anning Lin for providing JNK mutants.

      Supplementary Material

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